JPS58173553A - Serum purifying apparatus - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an adsorption purification device for malignant substances such as autoantibodies and immune complexes dissolved in plasma.

In recent years, autoimmune diseases such as cancer, immunoproliferative syndrome, rheumatoid arthritis, systemic lupus erythematosus, myasthenia gravis,
It has become clear that autoantibodies and immune complexes in plasma are deeply related to the causes and progression of nine diseases related to biological immune functions such as allergies and rejection reactions during organ transplantation.

The present inventors conducted extensive research on adsorbents that have high activity and adsorption selectivity for the above-mentioned malignant substances and can be safely used for body fluid purification treatment, and have discovered 1i15JJ1, a good adsorbent, and have previously filed a patent application (patent application). Showa 56-7152, Showa 56-
76776% 56-112919, 56-18.9
23, 56-159444).

In other words, it is an adsorbent in which a low molecular weight ligand is bound to an insoluble carrier, and it adsorbs autoantibodies and immune complexes at a high rate, has high adsorption selectivity, and can be easily sterilized by high-pressure steam sterilization. This makes safe and practical immunoadsorption therapy possible.

The present inventors have arrived at the present invention as a result of intensive research on the plasma purification device using the above-mentioned adsorbent. Namely, 1. The present invention consists of an insoluble carrier filled with an adsorbent in which a low molecular weight ligand is bound. A plasma purification device that purifies plasma using an adsorption column, which is characterized by having a means for passing the plasma to be purified at a flow rate such that the residence time in the column is at least 2 minutes. be.

Activated carbon and styrene resin are conventionally known as adsorbents for plasma purification, but both of these mainly target low molecular weight substances with a molecular weight of 1000 or less in plasma, such as uric acid, talleatinine, and bile acids. Therefore, the rate of diffusion of the substance was fast, and the method and conditions for passing the plasma did not matter. However, since autoantibodies and immune complexes, which are the targets of the present invention, are high molecular weight substances with molecular weights ranging from 160,000 to several million, their diffusion rate is slow, and this adsorbent is difficult to absorb between low-molecular ligands and malignant substances. Since affinity is used, the interaction force is relatively weak, so the adsorption method and conditions for high viscosity liquids such as plasma were completely unknown.

The present inventors filled a column with an adsorbent in which a low molecular weight Gant was bonded to an insoluble carrier, passed the plasma of patients with rheumatoid arthritis through the column at various flow rates, and evaluated the adsorption ability of rheumatoid factors in plasma. Surprisingly, when plasma is retained in the column, i5 is at least 2.
It has been found that 5 minutes are required, more preferably 5 minutes.

In the present invention, the residence time is an amount defined as the value obtained by dividing the void volume used in high performance liquid chromatography by the liquid passing rate.

Furthermore, when we repeatedly changed the column shape (L/f) and evaluated it, it became clear that it was uniquely determined by the residence time regardless of L/D (L/D0, 5 to 8). .

This is because the contact time between the whole adsorbent and the plasma is considered to be more important than the linear velocity of the plasma when adsorbing high molecular weight tannosic in plasma by this adsorbent, and the diffusion rate of the high molecular weight malignant substance is the main factor. It is understood that the carrier used in the present invention is a hydrophilic carrier. is preferably used because it minimizes nonspecific adsorption of plasma components. The shape of the carrier used is 5 spherical particles with a surface that has low resistance to blood flow through the plasma. As for the material of the carrier, known carriers such as dextran and agarose, which are usually used in affinity chromatography, can be used without any special requirements, but cross-linked polyvinyl alcohol can be particularly effectively used.
6-110817, 56-112919).

As described in the previous patent application, the low molecular weight ligand of the present invention is an organic low molecular compound with a molecular weight of 10,000 or less, an abanoic acid, a peptide, a nucleobase, a nucleoside, a nucleotide, a sugar, an oligosaccharide, IIjA phosphoric acid and the like can be exemplified.

The particle size of the adsorbent of the present invention is from 25 μm to 2500 μm.
can be used, preferably from 50 μm to 1
It is 500 μm.

When the purification device of the present invention is used for extracorporeal circulation therapy, the collected blood is separated into blood cell components and plasma components using a continuous centrifuge or a membrane-type plasma separator, and the plasma is transferred to the present column. This can be carried out by passing the liquid through a pump whose flow rate is controlled so that the residence time is at least 2 minutes, allowing the purified plasma and blood cell components to merge and returning the mixture. It can also be applied to intermittent plasma purification using a centrifuge.

The present invention can be applied not only to plasma but also to blood by converting the hematocrit value into the blood retention time.

Unexploded fIA defines an essential plasma purification device when applied to extracorporeal circulation therapy using adsorbent tubes that hold low molecular weight ligands, thereby making it possible to utilize the adsorption capacity of the adsorbent to its fullest extent. It is something.

The drawings are for explaining an example of the plasma purification device of the present invention. In the figure, the fluid is introduced into a plasma separator 1 by a disintegration pump P, where it is separated into blood cell components and plasma components. Blood cell components are on line 2.

The plasma components are respectively discharged into lines 5, and the plasma components flow through the pump P into the column 4 for plasma purification. The purified plasma passes through line 5 and joins the blood cell components. 6
is a regulator for adjusting the rotation speed of pump P2,
This makes it possible to adjust the true residence time in the column 4. In this example, the pump P and the regulator 6 pass the plasma at a plasma flow rate that makes the residence time in the column a predetermined value. constitutes a means.

Hereinafter, embodiments will be described with reference to Examples.

Example 1 Vinyl acetate 1002% triallyl isocyanurate 6
4,3f (X=0,40), ethyl acetate 100? , heptane 100t, polyvinyl acetate (polymerization degree 500) 7
．． 5 f and 2,7-azopisisoputilonitrile 3
．． 8t M-a mixture and polyvinyl alcohol 1
Weight%, dihydrogen phosphate) 17 um dihydrate 0.05
Heaviness and Disodium Hydrogen Phosphate Twelve Waters $1.5
Pour 400 ml of water in which the soybean paste was dissolved into a flask, stir thoroughly, and then heat at 5C for 18 hours.

Further, suspension polymerization was carried out by heating and stirring at 75°C for 5 hours to obtain a granular copolymer. After filtration, washing with water, and extraction with acetone, transesterification of the copolymer was carried out for 18 hours at 40 m in a solution consisting of 46.5 f of caustic soda and 2 t of methanol.

The average particle size of the obtained gel was 15.0 μm, and the vinyl alcohol unit (qOH) per unit weight was 9. Omeq
/r.

The specific surface area was 60 m''/7. The exclusion limit molecular weight with dextran was 6 x 10'. Next, the obtained gel 10f (dry weight) was suspended in dimethyl sulfoxide 12-, and epichlorohydrin 7B, Sad, 50% sodium hydroxide 10- was added and an activation reaction was carried out with stirring at 30C for 5 hours.

After the reaction, the mixture was washed with dimethyl sulfoxide, water, and dehydrated under suction. Next, this activated gel was mixed with tryptophan 1,6
0.1 M sodium carbonate buffer (pH 9) containing 3f
, 8) suspended in 160-. The immobilization reaction was carried out at 50C for 14 hours with stirring, and then 6Q, 6fw
Tris(hydroxyethyl)aminomethane solution of 55
- was added thereto, and a blocking reaction (to block remaining active groups) was carried out for an additional 50 hours with stirring. Thereafter, it was thoroughly washed with water to obtain an adsorbent for plasma purification treatment. The amount of tryptophan fixed on this adsorbent is 400μma
t/l (dry weight).

This adsorbent was packed into a column with an inner diameter of 10 mm and a length of 50 Iu, kept warm at S7C, and 9 μm CD-added lithiated patient plasma was passed through the column at a flow rate of about 6 mm.

Lithium factor (sensitized hemagglutination reaction R) before and after passing through the column
AHA test manufactured by Fuji Organs) and immune complex (C1q
binding method) was measured. The results are shown in Table 1.

Comparative Example 1 The same as Example 1 except that the residence time was 1 minute. The results are shown in Table 1.

Table 1 Example 2 Activated gel obtained in the same manner as in Example 1 (degree of crosslinking
0.4), phenyluanine was immobilized as a ligand in the same manner as in Example 1 to obtain an adsorbent for plasma purification treatment.

Phenylalanine immobilized on the adsorbent is 500μmo
t/f (dry weight).

Similar to Example 1, this adsorbent was subjected to an adsorption test on plasma of a patient suffering from rhymphoma. Rheumatoid factor and immune complex levels were measured before and after the passage. The results are shown in Table 2.

Table 2 Comparative Example 2 The rest was the same as in Example 2 except that the sounding time was 1 minute. The results are shown in Table 2.

[Brief explanation of drawings]

The drawing is an explanatory diagram of the plasma purification device of the present invention. 1...Plasma separator 2, 3...Line 4...Power 2m 5...Line 6...
...Regulator p,, p, ...Pump

Claims (1)

[Claims] In an apparatus for purifying plasma using an adsorption column packed with an adsorbent in which a low-molecular-weight ligand is bound to an insoluble carrier, the plasma to be purified is purified at a flow rate such that the in-column purification time is at least 2 minutes. A plasma purification device characterized by having a means for passing liquid.